The present invention relates to the modification of galactomannans present in green coffee beans by reducing the endogenous level of α-D-galactosidase activity. In particular the present invention pertains to a plant cell having a reduced α-D-galactosidase activity and to a plant harboring such a plant cell.
In coffee grains, cell wall polysaccharides account for approximately 48% of mature coffee bean dry weight, and of these, mannans represent approximately half. These polysaccharides are essentially insoluble in purified form and have very low galactose branching (Bradbury and Haliday, J. agric. Food Chem. 38 (1990), 389–392). Mannan polymers are acknowledged to be the main reason for the large losses of original green coffee weight encountered during preparation of soluble coffee drinks. The losses occur either when insoluble material remains as sediments during initial extraction or when precipitates and gels form during storage of coffee liquors. Mannans have also been shown to be the principal component responsible for cloudiness and precipitation during standing of coffee beverages.
In some plants, the degree of galactose branching on the mannan chains has been found to partially depend on the activity of the α-D-galactosidase (EC 3.2.1.22). This enzyme is capable of releasing α-1,6-linked galactose units from galactomannans stored in plant seed storage tissue or maturation (Buckeridge and Dietrich, Plant Sci. 117 (1996), 33–43). In addition, the accumulation of galactomannans having a very low galactose/mannose ratio in some plant endosperms or cotyledon tissues has been shown to correlate to peak α-D-galactosidase activity during maturation of these tissues and to the hardening and drying thereof (Kontos and Spyropoulos, Plant Physiol. Biochem. 34 (1996), 787–793).
α-D-galatosidases activity has also been associated with the capacity to remove galactose residues, i.e., α-1,6-linked to galactomannan polysaccharides, which brings about a decreased solubility of these polymers (McCleary, Carb. Res. 92 (1981), 269–285). Furthermore, the removal of galactose side chains from galactomannans seems to increase the capacity thereof to interact with other polysaccharides, e.g., xanthans in guar, with a concomitant formation of complex gel. Galactose branching on coffee grain mannans decreases from approximately 40% in young grains to the low level found in the mature grains during maturation. Concurrently, α-D-galactosidase enzyme activity increases during coffee grain maturation.
The coffee α-D-galactosidase cDNA has been cloned (Zhu and Goldstein, Gene 140 (1994), 227–231). According to the information derived therefrom, the mature coffee bean α-D-galactosidase is presumed to be composed of 363 amino acids and is synthesized as a pre-proenzyme of 420 residues. Following biosynthesis, two protease cleavages then remove a secretion signal (38 residues) and another signal peptide (19 residues) to produce the protein exhibiting the N-terminal amino acid sequence characteristic of the active enzyme.
In view of the known effects of galactomannans on the preparation and/or storability of soluble coffee, there is a need in the art to improve this situation. More specifically, there is a need to provide an improved method for preparing soluble coffee while concurrently obviating the drawbacks known when storing coffee liquors.